The human Mixed Lineage Leukemia protein-1 (MLL1) catalyzes histone H3 lysine 4 (H3K4) methylation, which is an epigenetic mark essential for the regulation of HOX genes in hematopoiesis and development. Translocations that disrupt the MLL1 gene are present in a unique group of acute leukemias, often predicting a poor prognosis. Other rearrangements and amplifications of MLL1 increase its enzymatic activity and are oncogenic. MLL1 contains an evolutionarily conserved SET domain (~130 amino acids) that catalyzes monomethylation of H3K4. Di- and possibly trimethylation of H3K4 require a conserved sub-complex that includes WDR5, RbBP5, Ash2L, and DPY-30 (WRAD). WRAD interacts with MLL1 forming what is known as the MLL1 core complex. Since different levels of H3K4 methylation are associated with different transcriptional outcomes, it is imperative to understand the molecular mechanisms by which the MLL1 core complex regulates the degree of H3K4 methylation. Despite the important biological role of MLL1 and its involvement in human leukemia, there is currently little information about the protein-structural features that ar responsible for its enzymatic activity. The long-term goal of this research is to fully characteriz the mechanisms responsible for the regulation of H3K4 methylation by the MLL1 core complex. This proposal takes a structure-function approach to investigate the molecular mechanisms underlying the multiple methylation activity of the human MLL1 core complex. We recently discovered that WRAD is a novel, non-SET domain histone methyltransferase that may catalyze H3K4 dimethylation within the MLL1 core complex. Here we propose to elucidate the mechanisms responsible for the regulation of H3K4 dimethylation by the MLL1 core complex in vitro and in vivo. To address these aims, a combination of molecular, biophysical and genome-wide approaches will be employed to investigate the regulation of H3K4 methylation by the MLL1 core complex. This information will increase our understanding of this key enzyme complex and its regulation in the pathways that control transcriptional activation in eukaryotes. This investigation is important because it may lead to better diagnostics and the rational design of anti-cancer drugs that inhibit MLL1's enzymatic activity.